Inhibitors of α1 β2 integrin mediated cell adhesion

ABSTRACT

The present invention relates to a compound of formula (I): wherein R is hydrogen atom, hydroxyl group or carbamoyl group, and n is 1 or 2, or a pharmaceutically acceptable salt thereof.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/US03/03449 which has an Internationalfiling date of Feb. 6, 2003, for which priority is claimed under 35U.S.C. §120. This application also claims priority to U.S. ProvisionalApplication No. 60/354,309, filed Feb. 7, 2002, under 35 U.S.C. §119(e).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds that are potentinhibitors of α_(L)β₂ integrin mediated cell adhesion which could beuseful for the treatment of α_(L)β₂ integrin mediated inflammatoryconditions.

2. Description of Related Art

Leukocyte integrins and intercellular adhesion molecules (ICAMs) playpivotal roles in leukocyte adhesion to target cells and extracellularmatrices. The β₂ (CD18) integrin subfamily has four members, eachconsisting of a related but distinct α-chain noncovalently paired withCD18: α_(L)β₂ integrin (LFA-1, CD11a/CD18), α_(M)β₂ integrin (Mac-1,CD11b/CD18), α_(X)β₂ integrin (p150/95, CD11c/CD18), and α_(D)β₂integrin (CD11d/CD18) (Bochner ed., Adhesion Molecules in AllergicDisease, Marcel Dekker, Inc. pp 1-24 (1997)). Among them, LFA-1 has beenshown to be central to the cell adhesion and transendothelial migrationof T cells, eosinophils, and other leukocytes into inflamed tissues(Garmberg, Curr. Opin. Cell Biology, 9, 643-650 (1997); Panes et al.,Br. J. Pharmacology, 126, 537-550 (1999)). LFA-1 binds to the ICAMfamily (ICAM-1, -2, -3, -4, -5) of molecules expressed on multiple celltypes such as vascular endothelial cells, dendritic cells, epithelialcells, macrophage and T lymphoblasts (Dustin et al., J. Immunology, 137,245-254 (1986)). In addition, LFA-1/ICAM-1 and LFA-1/ICAM-3 interactionscan act as co-stimulatory signals required for T cell activation(Wingren et al., Crit. Rev. in Immunology, 15, 235-253 (1995)).

Cell migration and T cell co-activation are important processes in anumber of inflammatory disease states. A dominant role of LFA-1 inmediating inflammatory events is shown in several different animalmodels of inflammatory diseases in which antibodies to LFA-1 or ICAM-1significantly inhibit development of therapeutic end points (Rothlein etal., Kidney International, 41, 617 (1992); Iigo et al., J. Immunology,147, 4167 (1991); Bennet et al., J. Pharmacol. and Exp. Therapeutics,280, 988 (1997)). In addition, a humanized monoclonal antibody to CD11a(the alpha chain of LFA-1) has shown efficacy in patients with psoriasis(Gottlieb et al., J. Am. Acad. Dermatology, 42, 428-35 (2000)).

Moreover, it has been shown that antibodies against LFA-1 suppressrejection after organ transplantation (Poston et al., Transplantation69, 2005-2013 (2000); Nakakura et al. Transplantation 62, 547-552(1996)). WO 94/04188 discloses the use of monoclonal antibodies directedagainst α_(L)β₂ integrin for all transplantations.

SUMMARY OF THE INVENTION

The present invention relates to novel compounds of formula (I):

wherein R is hydrogen atom, hydroxyl group, or carbamoyl group, and n is1 or 2, or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

The desired compounds of the present invention may exist in the form ofoptical isomers based on asymmetric atoms thereof, and the presentinvention also includes these optical isomers and mixtures thereof.

In an embodiment of the present invention, the steric configuration of abond need not be fixed. The compounds of the present invention may be acompound with a sole configuration or a mixture with several differentconfigurations.

In a preferred embodiment of the compounds of formula (I), R is hydrogenatom.

In another preferred embodiment of the compounds of formula (I), R ishydroxyl group.

In still another preferred embodiment of the compounds of formula (I), Ris carbamoyl group.

In a more preferred embodiment of the compounds of formula (I), n is 1.

In another more preferred embodiment of the compounds of formula (I), nis 2.

In a further preferred embodiment of the compounds of formula (I), R ishydrogen atom and n is 1.

In another further preferred embodiment of the compounds of formula (I),R is hydroxyl group and n is 1.

In still another further preferred embodiment of the compounds offormula (I), R is carbamoyl group and n is 2.

Most preferred compounds of the present invention are selected from thefollowing compounds:

-   (5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-acetylamino-1,3-diazabicyclo[3.3.0]octane-2,4-dione;-   (5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-hydroxyacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione;-   (5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-carbamoylpropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione.

The characteristic of the present compounds is the combination of theacylamino group at 7-position and the 4-cyanobenzyl group at 5-positionof 1,3-diazabicyclo[3.3.0]octane nucleus, where such characteristic isnot specifically described in prior publications.

The compounds of the present invention have potent inhibitory activityagainst both LFA-1 mediated cell adhesion and LFA-1 mediated T cellco-activation, and also show excellent bioavailability after oraladministration which reflects the overall improvement in (a) plasmaprotein binding, (b) aqueous solubility and (c) lipophilicity. Thecompounds of the present invention therefore show excellent in vivoefficacy against the unfavorable conditions caused by LFA-1 mediatedcell adhesion.

In addition, the compounds of the present invention have potentantagonistic activity on substance P receptor, i.e., Neurokinin 1 (NK1)receptor, as well. Substance P receptor antagonists are considered to beuseful for the treatment of inflammatory diseases such as asthma,rheumatoid arthritis, inflammatory bowel disease, cystitis, and othergastric disorders (Kraneveld et al., Int. Immunopharmacology, 1,1629-1650 (2001); Swain et al., Ann. Rep. Med. Chem., 34, 51-60 (1999);Ohnmacht Jr. et al., Ann. Rep. Med. Chem., 33, 71-80 (1998)). Thus thecompounds of the present invention has excellent therapeutic potentialagainst the unfavorable conditions caused or mediated by substance P.Also, the compounds of the present invention may show excellent effectson the treatment or prevention of inflammatory diseases due to the dualactivities of LFA-1 mediated cell adhesion inhibition and substance Preceptor antagonism.

Moreover, the compounds of formula (I) have reduced cytotoxicity and lowcytochrome P450 inhibitory activity as compared with those describedpreviously, and therefore, the compounds of the present invention mayhave reduced side effect potential.

The compounds of the present invention may be clinically used either ina free form or in the form of pharmaceutically acceptable salts thereof.Pharmaceutically acceptable salts include an acid-addition salt with aninorganic acid or an organic acid, and a salt with an inorganic base, anorganic base or an amino acid. Pharmaceutically acceptable salts alsoinclude an intramolecular salt thereof, or a solvate or hydrate thereof.

The compounds of the present invention may be formulated into apharmaceutical composition comprising a therapeutically effective amountof the compound as defined above and a pharmaceutically acceptablecarrier or diluent. The pharmaceutically acceptable carrier or diluentmay be, for example, binders (e.g., syrup, gum arabic, gelatin,sorbitol, tragacanth, polyvinylpyrrolidone), excipients (e.g., lactose,sucrose, corn starch, potassium phosphate, sorbitol, glycine),lubricants (e.g., magnesium stearate, talc, polyethylene glycol, silica)disintegrators (e.g., potato starch), wetting agents (e.g., sodiumlaurylsulfate), and the like.

The desired compounds of the present invention or pharmaceuticallyacceptable salts thereof may be administered either orally orparenterally, and it may be used as a suitable pharmaceuticalpreparation. These pharmaceutical preparations may be in the form of asolid preparation such as a tablet, a granule, a capsule, and a powder,or in the form of a liquid preparation such as solution, suspension, andemulsion, when administered orally. When administered parenterally, thepharmaceutical preparation may be in the form of suppository, aninjection preparation or an intravenous drip preparation using distilledwater for injection, a physiological salt solution, an aqueous glucosesolution, and so on, and an inhalation by a conventional process.

The dose of the desired compounds of the present invention orpharmaceutically acceptable salts thereof vary depending on anadministration method, age, sex, body weight, and condition of apatient, but, in general, the daily dose is preferably about 0.1 to 100mg/kg/day, particularly preferably 1 to 100 mg/kg/day.

The compounds of the present invention can be used for treating orpreventing LFA-1 mediated conditions in a patient, for example, a humanpatient. The compounds of the present invention can be also used for thetreatment of a patient suffering from or susceptible to LFA-1 mediatedconditions. Examples of LFA-1 mediated conditions include inflammatorydiseases, autoimmune diseases, and allergic diseases.

The compounds of the present invention can also be used for thetreatment or prevention of conditions caused or mediated by substance Pin a patient, as well as for the treatment of a patient suffering fromor susceptible to such conditions. Examples of the conditions may beinflammatory diseases.

The compounds of the present invention may be used for treatment orprevention of diseases such as rheumatoid arthritis, asthma, chronicobstructive pulmonary disease, allergy conditions, adult respiratorydistress syndrome, AIDS, cardiovascular diseases, thrombosis, harmfulplatelet aggregation, reocclusion following thrombolysis, reperfusioninjury, skin inflammatory diseases (e.g., psoriasis, eczema, contactdermatitis, atopic dermatitis), osteoporosis, osteoarthritis,atherosclerosis, arteriosclerosis including transplantation-associatedarteriosclerosis, neoplastic diseases including metastasis of neoplasticor cancerous growth, wound, detaching retina, Type I diabetes, multiplesclerosis, systemic lupus erythematosus (SLE), ophthalmic inflammatoryconditions, inflammatory bowel diseases (Crohn's disease and ulcerativecolitis), cystitis, gastric disorder, regional enteritis, Sjogren'sSyndrome, and other autoimmune diseases.

The compounds of the present invention may also be used for therejection (i.e., chronic rejection and acute ejection) after organtransplantation, including allograft rejection (host vs. graft disease)and graft vs. host disease.

The compounds of the present invention may be preferably used fortreatment or prevention of psoriasis, rheumatoid arthritis, inflammatorybowel diseases (Crohn's disease, ulcerative colitis), systemic lupuserythematosus, atopic dermatitis, Sjogren's syndrome, and rejectionafter organ transplantation (allograft rejection and graft vs. hostdisease).

The compounds of the present invention may be further preferably usedfor treatment or prevention of rheumatoid arthritis, asthma, chronicobstructive pulmonary disease, psoriasis, multiple sclerosis, andrejection after organ transplantation.

The compounds of the present invention may also be further preferablyused for treatment or prevention of inflammatory diseases such asasthma, inflammatory bowel disease, cystitis and other gastricdisorders.

According to the present invention, the desired compounds (I) can beprepared in accordance with one of the following methods:

Method A:

Among the compounds of the present invention, a compound of formula(I-a):

wherein n is the same as defined above, or a pharmaceutically acceptablesalt thereof, can be prepared by condensing a compound of formula (II):

or a salt thereof, with a compound of formula (III-a):H—(CH₂)_(n)—COOH  (III-a)wherein n is the same as defined above, a salt thereof, or a reactivederivative thereof, followed by converting the resulting compound into apharmaceutically acceptable salt thereof, if desired.

The salt of compounds (II) and (III-a) may be, for example, a salt withan inorganic or organic acid (e.g., trifluoroacetate, hydrochloride,sulfate), or a salt with an inorganic base (e.g., an alkali metal saltsuch as a sodium salt or a potassium salt, an alkaline earth metal saltsuch as a barium salt or calcium salt).

The condensation reaction of the compound (II) or a salt thereof withthe compound (III-a) or a salt thereof can be carried out in thepresence of a condensing reagent, with or without a base in a suitablesolvent.

The condensing reagent can be selected from conventional condensingreagents which can be used for a peptide synthesis, for example, BOP-Cl,BOP reagent, DCC, EDC or CDI. The condensing reagent can be preferablyused with an activator (e.g., HOBT).

The base can be selected from conventional organic bases such as analkylamine (e.g., DIEA, Et₃N), a cyclic amine (e.g., DBU, DBN,4-methylmorpholine), and pyridines (e.g., pyridine, DMAP), andconventional inorganic bases such as an alkali metal carbonate (e.g.,Na₂CO₃, K₂CO₃), an alkali metal hydrogen carbonate (e.g., NaHCO₃,KHCO₃), an alkali metal hydroxide (e.g., NaOH, KOH), and the like.

The solvent can be selected from any one which does not disturb thecondensation reaction, for example, esters (e.g., methyl acetate, ethylacetate), halogenoalkanes (e.g., CHCl₃, CH₂Cl₂), ethers (e.g., diethylether, THF, DME, dioxane), amides (e.g., DMF, N-methylpyrrolidone),ketones (e.g., acetone, methyl ethyl ketone), CH₃CN, DMSO, and H₂O, anda mixture of these solvents. The reaction can be carried out at atemperature of −50° C. to 50° C., preferably from 0° C. to roomtemperature.

The condensation reaction of compound (II) or a salt thereof with thereactive derivative of compound (III-a) is carried out in the presenceor absence of a base in a suitable solvent or without solvent.

Examples of the reactive derivative of the compound (III-a) are an acidhalide (e.g., an acid chloride), a reactive ester (e.g., an ester withp-nitrophenol), an anhydride thereof, a mixed anhydride with othercarboxylic acid (e.g., a mixed anhydride with isobutyric acid), and thelike.

The base can be selected from conventional organic bases such as analkylamine (e.g., DIEA, Et₃N), a cyclic amine (e.g., DBU, DBN,4-methylmorpholine), and pyridines (e.g., pyridine, DMAP), andconventional inorganic bases such as an alkali metal carbonate (e.g.,Na₂CO₃, K₂CO₃), an alkali metal hydrogen carbonate (e.g., NaHCO₃,KHCO₃), an alkali metal hydroxide (e.g., NaOH, KOH), and the like.

The solvent can be selected from any one which does not disturb thecondensation reaction, for example, esters (e.g., methyl acetate, ethylacetate), halogenoalkanes (e.g., CHCl₃, CH₂Cl₂), ethers (e.g., diethylether, THF, dioxane), amides (e.g., DMF, N-methylpyrrolidone), ketones(e.g., acetone, methyl ethyl ketone), CH₃CN, DMSO, and H₂O, and amixture of these solvents.

The condensation reaction can be carried out at a temperature of −30° C.to room temperature.

Method B:

Among the compounds of the present invention, a compound of formula(I-b):

wherein n is the same as defined above, or a pharmaceutically acceptablesalt thereof, can be prepared by condensing compound (II) or a saltthereof with a compound of formula (III-b):R¹O—(CH₂)_(n)—COOH  (III-b)wherein R¹O is a protected or unprotected hydroxyl group, and n is thesame as defined above, a salt thereof, or a reactive derivative thereof,followed by removing the protecting group, and further converting theresulting compound into a pharmaceutically acceptable salt thereof, ifnecessary.

The salt of compound (III-b) may be, for example, a salt with aninorganic base (e.g., an alkali metal salt such as a sodium salt and apotassium salt, and an alkaline earth metal salt such as a barium saltand a calcium salt).

The protecting group for the hydroxyl group can be selected fromconventional protecting groups for a hydroxyl group which can be easilyremoved by a conventional method. Examples of such protecting groupsinclude a trialkylsilyl group (e.g., trimethylsilyl group, triethylsilylgroup, and t-butyldimethylsilyl group), a benzyl group, a methyl groupand a tetrahydropyranyl group.

The condensation reaction of compound (II) or a salt thereof withcompound (III-b) wherein R¹O is a protected hydroxyl group, a saltthereof or a reactive derivative thereof can be carried out in a similarprocedure as described in Method A.

The removal of the protecting group can be carried out by a usual methodwhich is selected according to the protecting group to be removed, forexample, hydrolysis, acid treatment, BBr₃ treatment, and catalyticreduction.

The hydrolysis can be carried out by using an inorganic base such as analkali metal hydroxide (e.g., LiOH, NaOH, and KOH) in a suitable solventsuch as ethers (e.g., diethyl ether, dioxane, and THF), alcohols (e.g.,MeOH, EtOH), CH₃CN, DMSO, H₂O, and the like at room temperature or withheating.

The acid treatment can be carried out by using an inorganic acid or anorganic acid such as hydrochloride, hydrobromide, acetic acid,p-toluenesulfonic acid, and trifluoroacetic acid in a suitable solventsuch as ethers (e.g., diethyl ether, dioxane, THF), halogenoalkanes(e.g., CHCl₃, CH₂Cl₂), alcohols (e.g., MeOH, EtOH), CH₃CN, DMSO, H₂O,and the like at room temperature or with heating.

The catalytic reduction can be carried out by using a catalyst such aspalladium on activated carbon and Raney-nickel under a hydrogenatmosphere at room temperature or with heating in a suitable solventsuch as ethers (e.g., diethyl ether, dioxane, THF), esters (e.g., methylacetate, ethyl acetate), alcohols (e.g., MeOH, EtOH), CH₃CN, AcOH, H₂O,and the like.

The treatment with BBr₃ for the demethylation can be carried out in asuitable solvent (e.g., THF, CH₂Cl₂, AcOH) at a temperature of −78° C.to 50° C.

In case that compound (III-b) wherein R¹O is a hydroxyl group is usedfor the condensation reaction, the hydroxyl group of compound (III-b)should be protected in situ prior to the condensation reaction.

The protection of the hydroxyl group can be carried out by reactingcompound (III-b) with a trialkylsilyl halide in a suitable solvent withthe presence of a base. Examples of the trialkylsilyl halide includetrimethylsilyl chloride, triethylsilyl chloride, andt-butyldimethylsilyl chloride. The base can be selected fromconventional bases which are used for the hydroxyl group protection, forexample, triethylamine, imidazole, and pyridine. The solvent can beselected from any one which does not disturb the reaction, for example,esters (e.g., methyl acetate, ethyl acetate), aromatic hydrocarbons(e.g., benzene, toluene), halogenoalkanes (e.g., CHCl₃, CH₂Cl₂), ethers(e.g., diethyl ether, THF, DME, dioxane), amides (e.g., DMF,N-methylpyrrolidone), ketones (e.g., acetone, methyl ethyl ketone),CH₃CN, DMSO, and a mixture of these solvents. The reaction can becarried out at a temperature of −50° C. to 50° C., preferably from 0° C.to room temperature. The protected compound can be isolated in a usualprocedure, if necessary.

Method C:

Among the compounds of the present invention, a compound of formula(I-c):

wherein n is the same as defined above, or pharmaceutically acceptablesalt thereof, can be prepared by condensing compound (II) with acompound of formula (III-c):H₂NC(═O)—(CH₂)N—COOH  (III-c)wherein n is the same as defined above, a salt thereof, or a reactivederivative thereof, followed by converting the resulting compound into apharmaceutically acceptable salt thereof, if desired.

The salt of compounds (III-c) may be, for example, a salt with aninorganic base (e.g., an alkali metal salt such as a sodium salt or apotassium salt, an alkaline earth metal salt such as a barium salt or acalcium salt).

The reaction of compound (II) or a salt thereof with compound (III-c) ora salt thereof can be carried out in a similar method as described inMethod A.

The starting compound of formula (II) can be prepared in accordance withthe description of WO 01/30781 or the following scheme:

(In the above scheme, X is a C₁₋₆ alkyl group or a benzyl group, andtBDMSO is tert-butyldimethylsilyloxy group.)

-   Step (a): Compound (IV) can be prepared by reacting 4-hydroxyproline    C₁₋₆ alkyl or benzyl ester with 3,5-dichlorophenylisocyanate in the    presence of a base in a suitable solvent.

The base can be selected from conventional organic bases such as analkylamine (e.g., Et₃N, DIEA) and pyridine, and conventional inorganicbases such as an alkali metal hydrocarbonate (e.g., NaHCO₃, KHCO₃) andan alkali metal carbonate (e.g., Na₂CO₃, K₂CO₃).

The solvent can be selected from any one which does not disturb thecondensation reaction, for example, CH₂Cl₂, DME, THF, DMF, HMPA or amixture thereof. The reaction can be carried out at a temperature of−78° C. to room temperature.

-   Step (b): Compound (V) can be prepared by protecting the hydroxyl    group of compound (IV). The protection can be carried out by a usual    manner, for example, by reacting compound (IV) with    tert-butyldimethylsilyl chloride in the presence of imidazole in a    suitable solvent such as CH₃CN. The reaction is carried out at a    temperature of 0° C. to boiling point of the solvent, preferably at    room temperature.-   Step (c): Compound (VI) can be prepared by cyclizing compound (V).    The cyclization can be carried out in the presence or absence of a    base in a suitable solvent.

The base can be selected from conventional inorganic bases such as analkali metal alkoxide (e.g., NaOEt, NaOMe), an alkali metal carbonate(e.g., K₂CO₃, Na₂CO₃) and an alkali metal hydrocarbonate (e.g., NaHCO₃),and conventional organic bases such as pyridine, DMAP, Et₃N, and DIEA.

The solvent can be selected from any one which does not disturb thecyclization reaction, for example, toluene, DME, CH₂Cl₂, THF, CH₃CN,DMF, alcohols (e.g., MeOH, EtOH) or a mixture thereof. The reaction iscarried out at a temperature of 0° C. to boiling point of the solvent,preferably at 50° C. to 100° C.

-   Step (d): Compound (VII) can be prepared by condensing compound (VI)    with a compound of formula (XI):

wherein Y is a leaving group.

The leaving group may be selected from a halogen atom (e.g., a chlorineatom, a bromine atom, and an iodine atom), p-toluenesulfonyloxy group,and methanesulfonyloxy group.

The condensation reaction can be carried out in the presence of a basein a suitable solvent.

The base can be selected from conventional bases such as an alkali metalamide (e.g., LDA, KHMDS with or without LiCl).

The solvent can be selected from any one which does not disturb thecondensation reaction, for example, diethyl ether, DME, THF, DMF, HMPAor a mixture thereof. The reaction can be carried out at a temperatureof −78° C. to room temperature.

-   Step (e): Compound (VIII) can be prepared by deprotecting compound    (VII). The deprotection can be carried out by a usual method, for    example, treating the compound with HF/pyridine, n-Bu₄NF, or an acid    (e.g., HCl, AcOH, TFA, p-TsOH) in a suitable solvent or without a    solvent.

The solvent can be selected from any one which does not disturb thecondensation reaction, for example, CH₃CN, THF, DMF alcohols (e.g.,MeOH, EtOH) or a mixture thereof. The reaction can be carried out at atemperature of −78° C. to room temperature.

-   Step (f): Compound (IX) can be prepared by reacting compound (VIII)    with methanesulfonyl chloride in the presence of a base in a    suitable solvent.

The base can be selected from conventional bases such as Et₃N, DIEA,pyridine, NaHCO₃, KHCO₃, Na₂CO₃, K₂CO₃, and KHCO₃.

The solvent can be selected from any one which does not disturb thereaction, for example, CH₂Cl₂, THF, DMF, CH₃CN, toluene. The reactioncan be carried out at a temperature of −20° C. to 50° C.

-   Step (g): Compound (X) can be prepared by reacting compound (IX)    with an alkali metal azide (e.g., NaN₃).

The substitution reaction can be carried out at a temperature of 0° C.to 100° C. in an organic solvent.

The solvent can be selected from any one which does not disturb thereaction, for example, CH₂Cl₂, THF, DMF, CH₃CN, and toluene.

-   Step (h): Compound (II) can be prepared by reducing compound (X).    The reduction can be carried out under catalytic hydrogenation    conditions, for example, in the presence of a Pd or Pt catalyst    (e.g., Pd—C, PtO₂) in a suitable solvent under a H₂ atmosphere at    room temperature.

The solvent can be selected from any one which does not disturb thereaction, for example, EtOAc, MeOH, and EtOH.

In the present description and the claims, the C₁₋₆ alkyl group means astraight chain or branched chain alkyl group having 1 to 6 carbon atoms,for example, methyl group, ethyl group, propyl group, isopropyl group,butyl group, isobutyl group, etc., preferably one having 1 to 4 carbonatoms.

Abbreviations AcOEt: Ethyl acetate (=EtOAc) AcOH: Acetic acid BOP—Cl:Bis(2-oxo-3-oxazolidinyl)phosphinic chloride BOP reagent:Benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphateBSA: Bovine serum albumin CDI: Carbonyldiimidazole DBN:1,5-Diazabicyclo[4.3.0]non-5-ene DBU: 1,8-Diazabicyclo[5.4.0]undec-7-eneDCC: 1,3-Dicyclohexylcarbodiimide DIEA: Diisopropylethylamine DMAP:4-Dimethylaminopyridine DME: Dimethoxyethane DMF: Dimethyl formamideDMSO: Dimethyl sulfoxide EDC:1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride Et: EthylEtOH: Ethanol HBSS: Hank's balanced salt solution HMPA:Hexamethylphosphoramide HOBT: 1-Hydroxybenzotriazole hydrate HSA: Humanserum albumin KHMDS: Potassium hexamethyldisilazide (=Potassiumbis(trimethylsilyl)amide) LDA: Lithium diisopropylamide Me: Methyl MeOH:Methanol n-Bu: n-Butyl tBDMS: tert-Butyldimethylsilyl THF:Tetrahydrofuran TFA: Trifluoroacetic acid p-TsOH: p-toluenesulfonic acid

EXAMPLES

The compounds of the present invention are exemplified by the followingexamples but not limited thereby.

Example 1(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-acetylamino-1,3-diazabicyclo[3.3.0]octane-2,4-dione

To a solution of(5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione(78.5 mg) in THF (5 mL) was added acetic anhydride (1.0 mL). Thereaction mixture was stirred for 2 hours at 45° C., and the mixture wasconcentrated and purified by preparative thin-layer chromatography(silica gel; CH₂Cl₂) to afford the titled compound (84 mg). MS (m/z)478.8 (MNa⁺).

Example 2(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-carbamoylpropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione

A mixture of(5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione(82.7 mg), succinamic acid (45.86 mg), EDC (93.12 mg), HOBT (61.24 mg)and DIEA (104.79 μL) in THF (5 mL) was stirred overnight at roomtemperature. The reaction mixture was concentrated and purified by highperformance liquid chromatography (HPLC) (Beckman 5μ C18 column; elutedwith a gradient of H₂O/MeCN (10-100%)/0.1% TFA) to give 72 mg of thetitled compound. MS (m/z) 536 (MNa⁺).

Example 3(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-carbamoylacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione

A mixture of(5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione(200 mg), malonamic acid (59.5 mg), EDC (112 mg), HOBT (97.5 mg) andDIEA (168 μL) in THF (5 mL) was stirred overnight at room temperature.The reaction mixture was evaporated. The residue was dissolved in EtOAcand the resulting solution was washed with water, saturated aqueousNaHCO₃ solution, brine, dried (Na₂SO₄) and concentrated to give 212 mgof the titled compound. MS (m/z) 500 (MH⁺).

Example 4(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-hydroxypropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione

-   Step 1: A mixture of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (0.300 g), 3-methoxypropionic acid (0.209 μL), EDC (0.224 g), HOBT    (0.221 g) and DIEA (0.38 μL) in THF (15 mL) was stirred overnight at    room temperature. The reaction mixture was evaporated. The residue    was purified by HPLC [Beckman 5μ C18 column; eluted with a gradient    of H₂O/MeCN (10-100%)/0.1% AcOH] to give a foam. It was dissolved in    EtOAc and the resulting solution was washed with water, saturated    aqueous NaHCO₃ solution, brine, dried (Na₂SO₄) and concentrated to    give 0.259 g of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-methoxypropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    MS (m/z) 501 (MH⁺).-   Step 2: BBr₃ (3 mL, 1M in CH₂Cl₂) was added to a solution of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-methoxypropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (0.16 g) in CH₂Cl (15 mL) at −78° C. and the mixture was stirred for    8 hours at −78° C. The mixture was evaporated, and the residue was    purified by HPLC (Beckman 5μ C18 column; eluted with a gradient of    H₂O/MeCN (10-100%)/0.1% AcOH) to give foam. It was dissolved in    EtoAc and the resulting solution was washed with water, saturated    aqueous NaHCO₃ solution, brine, dried (Na₂SO₄) and concentrated to    give 0.119 g of the titled compound. MS (m/z) 487 (MH⁺).

Example 5(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-hydroxyacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione

-   Step 1: To a solution of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (150 mg) and DIEA (189 μL) in THF (4 mL) was added a solution of    benzyloxyacetyl chloride (57 μL) in THF (2 mL) and the mixture was    stirred overnight at room temperature. The reaction mixture was    concentrated and the residue was taken up in EtOAc. The resulting    solution was washed with brine, dried (Na₂SO₄), filtered and    concentrated. The residue was purified by HPLC (Beckman 5μ C18    column; eluted with a gradient of H₂O/MeCN (10-100%)/0.1% AcOH) to    give a foam. It was dissolved in EtOAc and the resulting solution    was washed with water, saturated aqueous NaHCO₃ solution, brine,    dried (Na₂SO₄) and concentrated to give 0 135 g of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-benzyloxyacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    MS (m/z) 563.4 [MH⁺].-   Step 2: Hydrogen was bubbled through a solution of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-benzyloxyacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione    from step 1 (0.125 g) in EtOH (10 mL) containing Pd/C (5%, 15 mg)    and the reaction mixture was stirred overnight under a H₂    atmosphere. The mixture was recharged with additional 5% Pd/C (10    mg) and stirred overnight under a H₂ atmosphere. The reaction    mixture was filtered through a bed of Celite and the filtrate was    concentrated. The residue was purified by HPLC (Beckman 5μ C18    column; eluted with a gradient of H₂O/MeCN (10-100%)/0.1% TFA) to    give 0.023 g of the titled compound. MS (m/z) 473 [MH⁺] and 495    [MNa+].

Reference Example 1(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione

The titled compound was prepared in accordance with the followingscheme:

(In the above scheme, tBDMSO is tert-butyldimethylsilyloxy group, andMsO is methanesulonyloxy group.)

-   Step-1: p-Toluene sulfonic acid (50.6 g) was added to a suspension    of L-4-trans-hydroxyproline (25.25 g) in a mixture of benzyl alcohol    (100 mL) and benzene (250 mL) and the mixture was heated under a    Dean Stark trap for 24 hours. The reaction mixture was concentrated    and diethyl ether was added to precipitate the solid. The solid was    filtered, washed with additional diethyl ether and dried to yield 75    g of L-4-trans-hydroxyproline benzyl ester.-   Step-2: To a suspension of L-4-trans-hydroxyproline benzyl ester    p-toluene sulfonic acid salt from step 1 (40.43 g) in THF (500 mL)    and DIEA (51.3 mL) was added 3,5-dichlorophenylisocyanate (22.1 g).    After stirring overnight, the reaction mixture was concentrated. The    residue was dissolved in EtOAc, washed with 0.5 N HCl, saturated    aqueous NaHCO₃ solution, brine, dried (Na₂SO₄), filtered and    concentrated. The residue was triturated in EtOAc/hexane (1:1) and    the white solid was filtered and purified via flash column    chromatography (silica gel; hexane/EtOAc 2:1) to yield    (2S,4R)-2-[(3,5-dichlorophenyl)carbamoyl]-4-hydroxyproline benzyl    ester (33.07 g).-   Step-3: To a suspension of    (2S,4R)-2-[(3,5-dichlorophenyl)carbamoyl]-4-hydroxyproline benzyl    ester (33.07 g) in CH₃CN (800 mL) was added imidazole (11 g) and    tert-butyldimethylsilyl chloride (13.64 g). After stirring for 48    hours, the reaction mixture was concentrated. The residue was    dissolved in EtOAc, washed with 0.5 N HCl, saturated aqueous NaHCO₃    solution, brine, dried (Na₂SO₄), filtered and concentrated. The    residue was purified via flash column chromatography (silica gel;    hexane/EtOAc 2:1) to give    (2S,4R)-2-[(3,5-dichlorophenyl)carbamoyl]-4-(tert-butyldimethylsilyloxy)proline    benzyl ester (44.45 g).-   Step-4: To a solution of the    (2S,4R)-2-[(3,5-dichlorophenyl)carbamoyl]-4-(tert-butyldimethylsilyloxy)proline    benzyl ester (23.49 g) in CH₃CN (500 mL) was added DIEA (34.44 mL)    and the mixture was heated to reflux. After refluxing for 24 hours    the reaction mixture was concentrated and purified by flash column    chromatography (silica gel; hexane to hexane/EtOAc 1:1) to separate    the two diastereomers of    3-(3,5-dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    Diastereomer A: 7.46 g, MS: m/z 415 (M⁺); and Diastereomer B: 10.66    g, MS: m/z 415 (M⁺).-   Step-5: The compound from step 4, diastereomer A or B, (12.73 g) was    benzylated as follows. n-Butyl lithium (30 mL, 1.6 M in hexane) was    added with stirring to a solution of diisopropylamine (6.5 mL) in    THF (100 mL) at −78° C. under a N₂ atmosphere. The mixture was    maintained at that temperature for additional 30 minutes. The    mixture was added via cannula to a solution of    3-(3,5-dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (12.73 g) in dry THF (100 mL) at −78° C. under a N₂ atmosphere.    After stirring at −78° C. for 30 minutes, 4-cyano-α-bromotoluene    (9.08 g) in THF (100 mL) was added. The reaction mixture was stirred    at −78° C. for 2.5 hours, then slowly warmed up to room temperature    and allowed to stand at room temperature for 0.5 hour. The reaction    mixture was concentrated and the residue was dissolved in EtOAc. The    EtOAc solution was washed with 0.5 N HCl, saturated aqueous NaHCO₃    solution, brine, dried (Na₂SO₄), filtered and concentrated. The    residue was purified via flash column chromatography (silica gel;    hexane/EtOAc 24:1 to 3:1) to give (5S,7R)- and    (5R,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione.-   (5S,7R) isomer: 7.6 g, MS: m/z 530 (M+); and-   (5R,7R) isomer: 1.8 g, MS: m/z 530 (M+)-   Step-6: To a solution of    (5S,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (1.0 g) in THF (1 mL) was added 70% HF/pyridine (25 mL). The    reaction mixture was stirred for 24 hours and then evaporated. The    residue was dissolved in EtOAc and the resulting solution was washed    with water, saturated aqueous NaHCO₃ solution, brine, dried (Na₂SO₄)    and concentrated. The residue was purified by flash column    chromatography (silica gel; MeOH/CH₂Cl₂ 2-7%) to give    (5S,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-hydroxy-1,3-diazabicyclo[3.3.0]octane-2,4-dione    (0.52 g). MS (m/z) 416 [MH⁺].-   Step 7: To a solution of    (5S,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-hydroxy-1,3-diazabicyclo[3.3.0]octane-2,4-dione    from step 6 (0.52 g) in CH₂Cl₂ (8 mL) at 0° C. was added DIEA (0.45    mL) followed by methanesulfonyl chloride (0.15 mL) and the mixture    stirred for 1.5 hours. The reaction mixture was diluted with CH₂Cl₂    and the resulting mixture was washed with saturated aqueous NaHCO₃    solution followed by brine, dried (Na₂SO₄), filtered and    concentrated to give 0.76 g of    (5S,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-methanesulfonyloxy-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    This compound was used as is for the next step. MS (m/z) 501 [MH⁺].-   Step 8: NaN₃ was added to a solution of    (5S,7R)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-methanesulfonyloxy-1,3-diazabicyclo[3.3.0]octane-2,4-dione    from step 7 (0.76 g) in DMF (5 mL) and the mixture was stirred for    24 hours. The reaction mixture was partitioned between EtOAc and    water. The organic solution was washed with brine, dried (Na₂SO₄),    filtered and concentrated. The residue was purified by flash column    chromatography (silica gel; CH₂Cl₂) to give 0.46 g of (5S,    7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-azido-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    MS (m/z) 441 [MH⁺].-   Step 9: Hydrogen was bubbled through a solution of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-azido-1,3-diazabicyclo[3.3.0]octane-2,4-dione    from step 8 (0.42 g) in EtOH (15 mL) containing Pd/C (5%, 15 mg) and    the reaction mixture was stirred overnight under a H₂ atmosphere.    The reaction mixture was filtered through a bed of Celite and the    filtrate was concentrated. The residue was purified by HPLC (Beckman    5μ C18 column; eluted with a gradient of H₂O/MeCN (10-100%)/0.1%    TFA) to give 0.21 g of    (5S,7S)-5-(4-cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione.    MS (m/z) 415 [MH⁺].

Reference Example 2(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-amino-1,3-diazabicyclo[3.3.0]octane-2,4-dione

-   Step 1:    3-(3,5-Dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione    was prepared by following similar procedures as described in    Reference Example 1, steps 1 through 4, but replacing    L-4-trans-hydroxyproline benzyl ester p-toluene sulfonic acid salt    with L-4-trans-hydroxyproline methyl ester hydrochloride.-   Step 2:    3-(3,5-Dichlorophenyl)-7-(tert-butyldimethylsilyloxy)-1,3-diazabicyclo[3.3.0]octane-2,4-dione    from step 1 was treated in similar procedures as described in    Reference Example 1, steps 5 through 9 to afford the titled    compound.    Cell Adhesion Protocol    Cell Adhesion The recombinant protein ICAM-1•Fc was constructed from    the 5 extracellular domains of human ICAM-1 and fusion with the    constant region of human IgG. ICAM-1•Fc was purified by Protein A    affinity chromatography and stored in aliquots at −20° C.    Immobilized ICAM-1•Fc was prepared by dilution of the protein in PBS    pH 7.5, transfer of 100 μl/well to Falcon Probind III plates and    overnight incubation at 4° C. Wells coated with BSA served as a    measure of non-specific background adhesion. Washed plates were    blocked with a solution of 0.25% ovalbumin in PBS for 1 h at 37° C.    HBSS washed Jurkat cells were suspended to a final concentration of    2.5×10⁶/ml in TBSg adhesion buffer (24 mM Tris pH 7.4, 0.14 M NaCl,    2.7 mM KCl, 2 mM glucose, 0.1% HSA). A 100 μl volume of cells was    added to the blocked and washed ICAM-1•Fc coated plates that    contained 100 μl of plate buffer (TBSg, 10 mM MgCl₂, 2% DMSO).    Adhesion was for 1 h at 37° C. Non-adherent cells were removed using    the EL404 plate washer (BioTek Instruments; Highland Park, Vt.). The    number of adherent cells was quantified by measuring enzymatic    activity of endogenous N-acetyl-hexosaminidase using the enzyme    substrate p-nitrophenol-N-acetyl-β-D-glucoseaminide, pNAG. The    amount of liberated p-nitrophenol was measured by reading the    optical density at 405 nm using a vertical pathway spectrophotometer    to quantify cell attachment (VMAX Kinetic Microplate Reader,    Molecular Devices, Menlo Park, Calif.). For competition studies the    compounds from 100% DMSO stock solutions were diluted in plate    buffer at 2-fold the required testing concentration prior to    transfer to the ICAM-1•Fc coated plate and serial dilution.

1. A compound of the formula (I):

wherein R is hydrogen atom, hydroxyl group or carbamoyl group, and n is1 or 2, or a pharmaceutically acceptable salt thereof.
 2. The compoundor salt according to claim 1, wherein R is hydrogen atom.
 3. Thecompound or salt according to claim 1, wherein R is hydroxyl group. 4.The compound or salt according to claim 1, wherein R is carbamoyl group.5. The compound or salt according to any one of claims 1-4, wherein nis
 1. 6. The compound or salt according to any one of claims 1-4,wherein n is
 2. 7. The compound or salt according to claim 1, wherein Ris hydrogen atom and n is
 1. 8. The compound or salt according to claim1, wherein R is hydroxyl group and n is
 1. 9. The compound or saltaccording to claim 1, wherein R is carbamoyl group and n is
 2. 10. Thecompound or salt according to claim 1, wherein the compound is selectedfrom the following compounds: (5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-acetylamino-1,3-diazabicyclo[3.3.0]octane-2,4-dione,(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(2-hydroxyacetyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione,(5S,7S)-5-(4-Cyanobenzyl)-3-(3,5-dichlorophenyl)-7-[(3-carbamoylpropionyl)amino]-1,3-diazabicyclo[3.3.0]octane-2,4-dione.11. A process for preparing a compound of formula (I-a):

wherein n is 1 or 2, or a pharmaceutically acceptable salt thereof,which comprises condensing a compound of formula (II):

or a salt thereof, with a compound of formula (III-a):H—(CH₂)_(n)—COOH  (III-a) wherein n is the same as defined above, a saltthereof or a reactive derivative thereof selected from the groupconsisting of an acid halide, a reactive ester, an anhydride thereof,and mixed anhydride with other carboxylic acid, followed by convertingthe resulting compound into a pharmaceutically acceptable salt thereof,if desired.
 12. A process for preparing a compound of formula (I-b):

wherein n is 1 or 2, or a pharmaceutically acceptable salt thereof,which comprises condensing a compound of formula (II):

or a salt thereof, with a compound of formula (III-b):R¹O—(CH₂)_(n)—COOH  (III-b) wherein R¹O is a protected or unprotectedhydroxyl group, and n is the same as defined above, a salt thereof or areactive derivative thereof selected from the group consisting of anacid halide, a reactive ester, an anhydride thereof, and mixed anhydridewith other carboxylic acid, followed by removing the protecting group,and further converting the resulting compound into a pharmaceuticallyacceptable salt thereof, if necessary.
 13. A process for preparing acompound of formula (I-c):

wherein n is 1 or 2, or a pharmaceutically acceptable salt thereof,which comprises condensing a compound of formula (II)

or a salt thereof, with a compound of formula (III-c):H₂NC(═O)—(CH₂)_(n)—COOH  (III-c) wherein n is the same as defined above,a salt thereof or a reactive derivative thereof selected from the groupconsisting of an acid halide, a reactive ester, an anhydride thereof,and mixed anhydride with other carboxylic acid, followed by convertingthe resulting compound into a pharmaceutically acceptable salt thereof,if desired.
 14. A pharmaceutical composition which comprises atherapeutically effective amount of the compound or salt as set forth inclaim 1 in admixture with a therapeutically acceptable carrier ordiluent.